[Comparative study of PCR-based Cryptosporidium discriminating techniques with a review of the literature]

Abstract

We identified the species or genotypes of the six Cryptosporidium isolates from patients and C. parvum strain HNJ-1 using the seven previously described species-differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. In addition, we also discussed about the usefulness of these PCR-based protocols on the basis of the reports previously published. Cryptosporidium diagnostic fragment was amplified by PCR with each primer pair, targeting the 18S ribosomal RNA (18SrRNA), Cryptosporidium oocyst wall protein (COWP). Heat shock protein 70 (HSP70), Polythreonine (Poly-T), Thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1), and unknown gene locus, in all isolates from patients and the strain HNJ-1. The RFLP profiles of 18SrRNA, COWP, HSP70, Poly-T, and TRAP-C1 PCR products in all isolates from patients were found to be the same among isolates, and were correspondent to those of C. parvum human genotype. While the RFLP profiles of HNJ-1 were strictly different from those of isolates from patients, and were correspondent to C. parvum cattle genotype. In addition, nucleotide sequences in 18 SrRNA gene of all isolates from patients and HNJ-1 were found to be identical to that of C. parvum, human or cattle genotype, respectively. Therefore, the isolates from patients and HNJ-1 were identified as C. parvum human and cattle genotype, respectively. According to the reports related to the PCR-based protocols applied in the present study, RFLP profiles targeting the HSP70, Poly-T, TRAP-C1 genes had been revealed in only a few species or genotypes, but those of 18SrRNA and COWP genes were in all species and genotypes. However, we supposed that it was difficult to distinguish between human or cattle genotype and other species or genotypes by RFLP profiles of 18SrRNA or COWP because the RFLP profiles of human or cattle genotype were identical or similar to those of other species or genotypes. On the other hand, it has been known that the nucleotide sequences in 18SrRNA or COWP gene are different among Cryptosporidium species and/or genotypes. Therefore, the direct sequencing method targeting the variable regions which can be used to distinguish among Cryptosporidium species, as well as the genotypes within C. parvum in either 18SrRNA or COWP gene is the most useful tool for accurate identification of Cryptosporidium isolates.